Background: Due to high tumor heterogeneity, breast cancer (BC) patients still suffer poor survival outcomes. YTHDF3 plays a critical role in the prognosis of BC patients. Hence, we aimed to construct a YTHDF3-based model for the prediction of the overall survival (OS) and the sensitivity of therapeutic agents in BC patients.Methods: Based on The Cancer Genome Atlas  database, we obtained BC patients’ data (n = 999) with YTHDF3 expression profiles. The association between YTHDF3 expression and 5-year OS was determined via Cox proportional hazards regression (CPHR) analysis. By integrating the variables, we established a prognostic nomogram. The model was estimated via discrimination, calibration ability, and decision curve analysis (DCA). The performance of the model was compared with the TNM stage system through receiver operating characteristic (ROC) curves and DCA. By means of the Genomics of Drug Sensitivity in Cancer (GDSC) database (https://www.cancerrxgene.org/), the therapeutic agents’ response was estimated. Gene set enrichment analysis (GSEA) demonstrated possible biological mechanisms related to YTHDF3. TIMER and CIBERSORTx were employed to analyze the association between YTHDF3 and tumor-infiltrating immune cells.Results: The high YTHDF3 expression was significantly correlated with poor 5-year OS in BC patients. Through multivariate CPHR, four independent prognostic variables  were determined. On the basis of the four factors, a YTHDF3-based nomogram was built. The area under the curve (AUC) of the ROC curve for the model surpassed that of the TNM stage system . The model predictions showed close consistency with the actual observations via the calibration plot. Therapeutic response prediction was conducted in high- and low-risk groups and compared with each other. The BC patients with higher risk scores showed more therapeutic resistance than those with a lower risk score.Conclusion: YTHDF3 was verified as a prognostic biomarker of BC, and a novel YTHDF3-based model was constructed to predict the 5-year OS of BC patients. Our model could be applied to effectively predict the therapeutic response of commonly used agents for BC patients. Breast cancer (BC) has surpassed lung cancer and has become the most commonly diagnosed tumor in 2020 . In ligh6-methyladenosine (m6A) is a noted posttranscriptional modification of coding RNAs and noncoding RNAs (ncRNAs)  . m6A can(ncRNAs) , and pla(ncRNAs) . Accumul(ncRNAs) . m6A mod(ncRNAs) . In ligh(ncRNAs) . They unPrevious studies have demonstrated the effectiveness of immunotherapy in BC patients. Thus, the immune checkpoint inhibitors were applied in the clinic . NeverthHerein, our study aimed to explore the YTHDF3 expression with the prognosis of BC patients, as well as the immune infiltrates. Furthermore, we attempted to establish a YTHDF3-based nomogram to assess the individual risk of 5-year OS in BC patients. Based on the clinical tool, we sought to predict the therapeutic responses of commonly used drugs in BC patients.Level 3 RNA sequencing profiles of BC patients and accompanying clinical characteristic data were obtained from TCGA database. We excluded the samples which had absent or deficient information on age, TNM stage, overall survival (OS) time, or survival status. The inclusion criteria were as follows: 1) local invasive BC identified by using the pathological method; 2) OS time was longer than 1 month; and 3) both survival data and RNA expression profiles were completely available. Clinical features contained TNM stage, T stage, N stage, age, estrogen receptor (ER), progesterone receptor (PR), HER-2 status, tumor subtypes, OS, and OS time. Thus, 999 BC patients were divided into two groups (the high- and the low-expression groups) depending on the median value of YTHDF3 expression. The correlation between YTHDF3 expression and the clinicopathological factors was determined. Meanwhile, we analyzed the YTHDF3 differential expression in 27 diverse kinds of cancers.https://cistrome.shinyapps.io/timer). We performed the YTHDF3 expression in BC with the abundance of tumor-infiltrating immune cells (TIICs) through gene modules in TIMER. Furthermore, by using CIBERSORTx (http://cibersortx. Stanford. edu/), which is a deconvolution algorithm dependent on gene expression, we analyzed the immune microenvironment to explore the correlation between TIICs and YTHDF3 expression in BC patients.The Tumor Immune Estimation Resource (TIMER) database provided a comprehensive analysis of immune infiltrates of various types of tumors  q < 0.25 were the significant threshold.In our report, 999 BC cases were divided into two groups (the high- and low-expression groups) according to the median value of YTHDF3 expression. We performed GSEA to determine the biological process that is enriched by YTHDF3 in BC patients. We selected “c2.cp.kegg.v6.2.symbols.gmt” as the gene set. The permutation number was set at 1,000 times, the minimum and maximum number sizes for gene analysis at 15 and 1,000, respectively. Gene sets with a nominal via a calibration curve. In addition, DCA was used to illustrate the clinical utility of the model.According to the analysis of univariate and multivariate CPHR, the YTHDF3-based model was conducted. The performance of the nomogram was quantified in reference to the ability of discrimination, calibration, and clinical utility. The score test was applied to estimate the goodness of fit of the model. The AUC of ROC curves was utilized to figure out the discrimination ability. The calibration ability was assessed https://www.cancerrxgene.org)  . Five covia the TIMER and CIBERSORTx algorithm. R software (version 4.0.3) was applied to determine the correlation between the expression of the RNA-seq gene and clinical parameters in BC. A p value <0.05 meant a significant difference.The Wilcoxon rank-sum test was applied to compare the expression of YTHDF3 in BC and normal groups. We used the Kaplan–Meier (K-M) method and log-rank test to analyze the OS of two groups (the high- and low-YTHDF3 expression groups), respectively. CPHR analysis was conducted to screen the independent factors of OS. Ridge regression analysis was performed to estimate the value of IC 50. The association between YTHDF3 and TILs was determined The clinical characteristics of 999 BC patients, including age, TNM stage, molecular subtypes, ER, PR, HER-2, and vital status, were extracted from TCGA database. In light of the inclusion and exclusion criteria, 999 patients were involved in later analysis. In our research, the patients’ age when diagnosed ranged from 26–89 years, and the medium of age was 57–91 years. The median follow-up time was 42.4 months, with median ranged from 1 to 286.8 months. The result was shown in p < 0.001), as well as in 21 of the 27 analyzed cancer types , age , and TNM stage III are significantly associated with OS. Multivariate CPHR uncovered that YTHDF3 expression was an independent prognostic factor for OS. The expression distribution and profiles of YTHDF3 and survival status of BC patients are demonstrated in We conducted the CPHR analysis to figure out the correlation between OS and the YTHDF3 expression, as well as other clinical characteristics in BC patients. As p = 0.013).In order to determine the prognostic role of YTHDF3 in BC, the expression data were separated into high- and low-risk groups in light of the median value of YTHDF3 expression. The K-M method was conducted to describe the OS of two groups. The log-rank test of OS curves showed that elevated YTHDF3 expression was combined with shorter OS time in BC patients (via TIMER. As shown in p = 5.02 × 10–3), CD4+ T cells (p = 1.56 × 10–3), CD8+ T cells (p = 1.11 × 10–31), macrophages (p = 8.96 × 10–24), neutrophils (p = 9.98 × 10–13), and dendritic cells (p = 1.16 × 10–7). These results revealed that YTHDF3 was a crucial part in the tumor immune infiltration of BC.The tumor-infiltrating lymphocytes (TILs) can independently predict the survival status . Furtherhttps://cibersortx.stanford.edu/) was utilized to identify different infiltrating levels of diverse immune cell subtypes in the high- and low-YTHDF3 expression groups , plasma cells (p = 0.017), activated NK cells (p < 0.001), regulatory T cells (p < 0.001), and M2 macrophages (p = 0.004) apparently increased in low-YTHDF3 expression group. In contrast, CD8+ T cells (p < 0.001), resting memory CD4+ T cells (p < 0.001), resting mast cells (p = 0.003), and activated memory CD4+ T cells (p = 0.008) were significantly elevated in the high-YTHDF3 expression group.Meanwhile, we attempted to determine if the immune infiltrating microenvironment varied according to the high and low YTHDF3 expression levels. The CIBERSORTx algorithm  were significantly differentially enriched in the high-YTHDF3 expression group, including ubiquitin-mediated proteolysis, inositol phosphate metabolism, epithelial cell signaling in Helicobacter pylori infection, and glycosylphosphatidylinositol GPI anchor biosynthesis, whereas the gene set involved in the low-YTHDF3 expression group was glycosaminoglycan biosynthesis chondroitin sulfate and ribosome. All snapshots of enrichment results are shown in GSEA was performed to select the YTHDF3-related signaling pathway in BC. In light of the median YTHDF3 expression value, TCGA data were divided into the high- and low-YTHDF3 expression groups. A nominal p value was 5e-13, which indicated good model fitness. The AUC of the model was 0.721 (95% CI: 0.654–0.789), which suggested good recognition ability of the model and prediction superiority over the TNM staging system   . The cal0.00028) . Further were selected as the candidates, which might show drug resistance in the high-risk group Our study demonstrated that higher YTHDF3 expression meant worse OS and was associated with lower TIL abundance. Based on the results of multi-CPHR, we built a YTHDF3-based nomogram combined with other clinical features. The model demonstrated superior prognostic accuracy and better clinical values than the TNM stage. Finally, we performed therapeutic response prediction of BC patients in the high- and low-risk groups. The BC patients with high-risk scores were predicted to be less sensitive to several commonly used therapeutic agents, including doxorubicin, paclitaxel, methotrexate, and vinorelbine.Several studies had proposed nomograms to predict OS in BC patients. Zhao et al. reported that the models incorporating age, tumor stage, and molecular subtype could predict the OS in BC patients, respectively. However, all those models omitted the predictive value of genes associated with m6A modification. As mentioned in prior research studies, YTHDF3 efficiently improved the translational process of its targets and depended on m6A methylation . YTHDF3 In order to predict the drug sensitivity, several commonly used cytotoxic drugs and the small-molecule inhibitor lapatinib were screened out. The estimated IC50 values of these drugs were remarkably increased in the high-risk group. The higher IC50 values meant that the BC patients demonstrated less susceptibility to the agents they have taken. Anthracyclines and paclitaxel play fundamental roles in BC chemotherapy. Nevertheless, due to the high rate of chemoresistance, the survival outcome of BC patients is unsatisfactory. If oncologists were available to recognize the worthless drugs before making a prescription using a tool, the heavy medical burden on patients and society may be reduced. Fortunately, this study proposed a novel nomogram based on YTHDF3 expression, which was valuable in guiding the selection of therapeutic agents. Furthermore, it had the capability of picking out those who might be the potential ineffective candidates.Equally important, we employed the TIMER database to reveal connections between immune infiltration levels and YTHDF3 expression in BC. The associations of YTHDF3 expression with CD8+ T cells, dendritic cells, neutrophils, and macrophages were the strongest. Meanwhile, NK cells, macrophages, and mast cells showed a moderate to strong positive linkage with YTHDF3 expression. According to the CIBERSORTx result, the infiltration levels of plasma cells, Treg cells, activated NK cells, and M2 macrophages were decreased in the high-YTHDF3 expression group compared with the low-expression one. NK cells are cytotoxic and secrete cytokines and cytotoxic granules, which could suppress the cancer cell proliferation and mediate the antitumor process of NK cells . Muntasell et al. showed that NK cell infiltration is associated with anti-HER-2 treatment in HER-2-positive BC patients . Overexpression of YTHDF3 might inhibit the function and efficiency of NK cells in antitumor immune response, which could be one of the routes that YTHDF3 affects the tumor immune. The closed correlation implies a likely mechanism by which YTHDF3 regulates the biological functions of tumor-infiltrating immune cells in BC patients.Several limitations can be mentioned in our research. First, the role YTHDF3 played in therapeutic agents’ resistance and the potential molecular mechanism should be further investigated using additional experiments. Second, a different population from another center or dataset is needed to externally validate the prediction nomogram. Finally, we could not assess the influence of postoperative information (chemoradiotherapy and endocrine therapy) due to the unavailability of data from TCGA database.In sum, we confirmed the significant association between YTHDF3 expression and the 5-year OS in BC patients. Then, a novel nomogram was efficiently conducted to predict 5-year OS by integrating YTHDF3 expression, age, molecular subtype, and TNM stage. Our model can be utilized to predict the sensitivity of therapeutic agents and guide oncologists to select the appropriate therapeutic regimens.


ZFPM2-AS1 and MIR210HG to predict the hepatocellular carcinoma (HCC) prognosis and further explore the clinical significance of these lncRNAs, and the robustness of the model was validated in the verification set. Thereafter, we calculated the immune score for each patient and explored the relationship between genome instability and the immune microenvironment. The analysis indicated that this model was better than the immune microenvironment in predicting the prognosis of HCC patients, suggesting that the GLncM may be an effective indicator of HCC prognosis and providing a new direction and strategy for estimating the prognosis of HCC patients.Genomic instability is a characteristic of tumors, and recent studies have shown that it is related to a poor prognosis of multiple cancers. Long non-coding RNAs (lncRNAs) have become a research hotspot in recent years, and many unknown biological functions are being explored. For example, some lncRNAs play a critical role in the initiation and progression of multiple cancer types by modulating genomic instability. However, the role of genomic instability-related lncRNAs in liver cancer remains unclear. Therefore, we screened genomic instability-related lncRNAs by combining somatic mutation data and RNA-Seq data in The Cancer Genome Atlas (TCGA) database. We established a genomic instability-related lncRNA model (GLncM) involving  Primary liver cancer is the sixth most commonly diagnosed cancer worldwide, and its mortality rate has increased to third . PrimaryGenomic instability is a prevalent characteristic of most cancers, from precancerous lesions to advanced tumors . In HCC,Long non-coding RNAs (lncRNAs) are a class of non-protein-coding RNA molecules whose transcripts are more than 200 nucleotides long . To dateIn this article, we combined genetic instability with lncRNAs, classified patients in The Cancer Genome Atlas (TCGA)-LIHC database according to lncRNA expression levels and somatic mutations, and analyzed clinical characteristics to construct an HCC prognostic risk model.n = 90) were selected as the genomic stable group, and the bottom 25% (n = 93) were selected as the genomic unstable group. The number of mutations in the genomic stable group ranged from 3 to 78, and the number of mutations in the genomic unstable group ranged from 154 to 2055. Then, we performed a differential expression analysis using the Wilcoxon rank-sum test. As shown in versus 102, p < 0.001, Wilcoxon rank-sum test, n = 153), and the group with a lower number of mutations was identified as the genomic stable-like (GS-like) cluster (n = 221).The workflow describing the study is shown in We established a ceRNA network based on the lncRNA expression profiles, mRNA expression profiles and microRNA expression data and visualized the network using the ggalluvial R package (Version: 0.12.3) to better understand the function of 88 genomic instability-related lncRNAs . Seventyn = 186) and verification set (n = 184) at a 1:1 ratio. The clinical features of the training set and verification set are displayed in p < 0.05; ZFPM2-AS1 and MIR210HG, p < 0.05, ZFPM2-AS1 and MIR210HG to evaluate the prognosis of HCC patients. The formula was as follows: GLncM score = 0.119315765508808 × expression level of ZFPM2-AS1+0.142471464922762 × expression level of MIR210HG. In the formula, the coefficients of ZFPM2-AS1 and MIR210HG were both positive, indicating that these two genes were risk factors and that high expression of these genes was correlated with a poorer prognosis than low expression. Liu et al. and Wang et al. previously confirmed that high expression of the two lncRNAs was associated with the proliferation, migration and invasion of HCC cell lines. According to the previous ceRNA network, microRNAs that interact with ZFPM2-AS1 and MIR210HG play a pivotal role in oncogenesis and tumor development , and the hazard ratio (HR) of the OS of low-versus high-risk group was 0.394 (95% CI: 0.223-0.696). The area under the receiver operating characteristic (ROC) curve (AUC) of the GLncM for OS was 0.742 (95% CI: 0.652-0.833) at 1 year, 0.741 (95% CI: 0.639-0.844) at 3 years and 0.654 (95% CI: 0.52-0.787) at 5 years  was regarded as the cutoff value to classify the samples into a high-risk group and a low-risk group. As presented in  5 years . In addining set .p = 0.016, ZFPM2-AS1 and MIR210HG expression patterns and the landscape of mutation counts and risk scores in the testing set  and the non-HBV-affected group (n = 93). According to the GLncM and cutoff value identified previously, patients in each HBV infection status group were divided into high- and low-risk subgroups. As described in p = 0.035), and similar results were observed in the non-HBV-affected group , and those with stage III or IV tumors were grouped into stage III - IV (n = 90). The GLncM with the cutoff value acquired previously divided the patients in the stage I–II group into a high-risk subgroup (n = 126) and a low-risk subgroup (n = 128), and the high-risk patients experienced a shorter OS than the low-risk patients  and a low-risk subgroup (n = 32), and a significant difference in OS was observed between the two groups  infection status, non-alcoholic fatty liver disease, alcohol consumption, pathologic stage and histologic grade, were subjected to a univariate Cox regression analysis and multivariate Cox regression analyses with the established GLncM to assess whether the GLncM was independent of common clinical features. As shown in We further assessed the predictive performance of GLncM by comparing it with two existing lncRNA-related models using the whole TCGA-LIHC dataset: the 5 lncRNA-based model from the study the study by Hou (HouLncM)  and the p = 0.004) compared to HouLncM (p = 0.034) compared to SunLncM (As shown in  HouLncM  and incr SunLncM .p < 0.001). Then, we analyzed the distribution of immune scores among the different risk groups. Patients in the high-risk group showed a significantly lower immune score than patients in the low-risk group  , we usedp = 0.032, p = 0.0065, p = 0.024, p = 0.055, p = 0.00036, Afterward, we analyzed the samples from the training set and verification set. Consistent with the results from the whole TCGA-LIHC dataset, patients in the high immune score group from the training set experienced a longer OS than those in the low immune score group . ZFPM2-AS1 and MIR210HG have previously been reported to be involved in cell proliferation and migration  database, we did not use the external dataset to verify the model. More prospective datasets are needed for the validation and assessment of the performance of the model to ensure its robustness and reproducibility.Data were acquired from TCGA database; these data included somatic mutation data for 364 primary HCC samples, microRNA expression profiles for 375 primary HCC samples, and gene fragments per kilobase of transcript per million reads mapped (FPKM) expression profiles and clinical characteristics for 374 primary HCC samples. In the modeling process, only 370 samples with both mRNA expression data and clinical data were used for analysis. We annotated the gene symbols and gene types according to the Homo_sapiens.GRCh38.102. chr.gtf file.We used a method similar to that described by Bao et al. to determine the lncRNAs associated with genomic instability . First, LncRNA expression data, mRNA expression data and microRNA expression data were used to construct the ceRNA network, and the network was visualized using the ggalluvial R package (Version: 0.12.3) to better illustrate the functions of lncRNAs associated with genomic instability.The construction of the ceRNA network mainly included two steps. 1) The differentially expressed mRNAs and microRNAs between the genomic stable group and genomic unstable group were obtained using the Wilcoxon rank-sum test and edgeR package (Version: 3.24.3) , respectp < 0.05 were screened in a subsequent multivariate Cox regression analysis. The lncRNAs with p < 0.05 in the multivariate Cox regression analysis were regarded as the final lncRNAs for model construction. Genomic instability-related lncRNAs were obtained by performing univariate and multivariate Cox regression analyses using the survival R package (Version: 3.1–12). The predictive power of the prognostic risk model was evaluated using the log-rank test and Kaplan‒Meier survival analysis.First, HCC samples were randomly split into a training set and verification set. Then, in the training set, the prognostic value of genomic instability-related lncRNAs for OS was estimated by performing a univariate Cox regression analysis. The lncRNAs with CIBERSORT was used


To propose a new definition of partial remission (PR) for patients with type 1 diabetes (T1D) of all-ages using insulin dose and glycated albumin (GA), and find the optimal cut-off values for stimulated C-peptide to diagnose PR in different age-groups.Patients with newly diagnosed T1D (n=301) were included. GA/insulin dose was used to diagnose PR, and insulin dose-adjusted glycated albumin (IDAGA) was proposed to facilitate clinical application. The optimal diagnostic levels of IDAGA and stimulated C-peptide were determined in different age-groups . Furthermore, the diagnostic consistency between different PR definitions was studied.GA≤ 23%/insulin dose ≤ 0.5u/kg/day was used to define PR, and IDAGA (GA (%) + 40 * insulin dose(u/kg/day)) ≤ 40 was feasible in all age-groups. Whereas, the optimal diagnostic level showed difference for stimulated C-peptide . About 40% of patients met the PR definition by stimulated C-peptide but not GA/insulin dose or IDAGA, who showed dyslipidemia and higher insulin resistance.A new definition of the PR phase is proposed using GA/insulin dose, and the calculated IDAGA≤ 40 applies to all age-groups. The stimulated C-peptide to diagnose PR is the highest in the 12-18y age-group, which reflects the effect of puberty on metabolism. For patients with insulin resistance, it is not recommended to use stimulated C-peptide alone to diagnose PR. Type 1 diabetes (T1D) is a chronic disease characterized by persistent autoimmune destruction of β cells, leading to lifelong dependence on exogenous insulin . The impThe following are the three definitions of PR frequently applied in clinical practice : 1) low Therefore, our study aims to propose a new definition of the PR phase using GA and insulin dose, and verify whether the diagnostic criteria of PR by IDAGA and stimulated C-peptide are suitable for different age-groups.From October 2015 to December 2020, 1328 patients from the T1D cohort at the Second Xiangya Hospital of Central South University were screened . This cohort has been described in detail in previous studies . ParticiThe baseline characteristics recorded include: gender, age of onset, duration of diabetes, diabetic ketosis/ketoacidosis at onset, waist circumference, blood pressure, hip circumference, daily insulin dosage (U/kg/day), HbA1c, glycated albumin (GA), serum lipid, fasting C-peptide (FCP), stimulated C-peptide, islet autoantibodies.The patients were followed up at an interval of 3-6 months, and the clinical information collected at each follow-up includes: age, duration of diabetes, waist circumference, hip circumference, blood pressure, daily insulin dosage, HbA1c, GA, serum lipid, FCP, and stimulated C-peptide after mixed meal tolerance test (MMTT).HbA1c was measured by automated liquid chromatography . GA was tested by the enzymatic method using Lucica Glycated Albumin‐L  as the measuring reagent and Mindray BS240  as the measuring instrument. Total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were determined by a Hitachi 7170 analyzer . GADA, IA-2A, and ZnT8A were detected by radioimmunoassay as previously reported . The cutA standard 543.6 kcal MMTT, with 44.4% carbohydrates, 47.7% fat and 7.9% protein, were performed before 10:00 am after an overnight fast. Long-acting insulin was used normally on the day before the MMTT, but short-acting insulin after breakfast on the test day was withheld. The MMTT was conducted with the fasting glucose levels between 3.9 and 11.1 mmol/L (70 and 200 mg/dL) and at least two weeks after the correction of ketoacidosis. Serum C-peptide levels were assessed at 0  and 120 minutes (stimulated C-peptide) after the first bite of a mixed meal. C-peptide levels were measured by a chemiluminescence method using the Advia Centaur System kit .Insulin resistance was measured by the estimated glucose disposal rate (eGDR) based on the following equation: eGDR (mg/kg/min) = 24.31-(12.22×waist to hip ratio)--(0.57×HbA1c), where the hypertension status was defined as blood pressure higher than 140/90 mmHg  curves. We calculated that a sample size of 62  patients would provide a statistical power of 0.8 to find the optimal cut-off point for PR diagnosis  at an alpha level of 0.05 (two-sided) on the basis of a one-sample ROC curve . After the enrollment process, 301 eligible patients were included, which fulfilled the calculated sample size.Data are presented as the mean ± standard deviation (SD) or median (25th percentile-75th percentile), or according to the specific notes in the article. Spearman′s rank correlation and simple linear regression were used to determine the association between HbA1c and GA so as to find the cut-off value for GA to define PR. Logistic regression was used to calculate the regression coefficient of GA and insulin dose. When comparing the stimulated C-peptide levels of patients in PR and not in PR, multiple follow-up points for the same patient were included for analysis and generalized estimation equation (GEE) was performed for these repeated- measures data. When comparing clinical characteristics between patients who met the criteria of PR by stimulated C-peptide but not GA/insulin or IDAGA and by both stimulated C-peptide and GA/insulin or IDAGA, the follow-up points where patients first met the criteria above were included, and student’s t test was carried out for variables with the normal distribution, while Mann-Whitney U test was used for data with non-normal distribution. SPSS 25.0 was used for the analysis above. The optimal cut-off point was determined by receiver operator characteristic (ROC) curve and restricted cubic spline (RCS). ROC was employed to find the optimal cut-off point of stimulated C-peptide and IDAGA to define the PR in different age-groups, with the GA/insulin dose being the diagnostic criteria. In addition, RCS was used to verify these optimal cut-off points obtained from ROC. The analysis was done using R version 4.0.5. Statistical significance was considered at P < 0.05.A total of 301 patients with new-onset T1D  were enrolled in our study, all of whom were positive for at least one islet autoantibodies Figure 1GA was positively correlated with HbA1c , with GA of 23.4% corresponding to HbA1c of 7.5%. As HbA1c ≤ 7.5% in combination with an insulin requirement of ≤0.5u/kg/day was commonly used to define the PR phase, PR was defined as GA ≤ 23.0% and an insulin requirement of ≤ 0.5u/kg/day in our study.In order to make the new PR definition practical to use in the clinic, logistic regression was performed to transform the definition into a quantitative measure. During the analysis, GA and insulin dose were taken as independent variables, and the PR diagnosis was the dependent variable. The regression coefficient was -20.5 for insulin dose and -0.5 for GA. As the ratio was roughly 40 between the two independent variables, IDAGA = GA (%) + 40 * insulin dose (u/kg/day) was proposed. With GA/insulin dose being the diagnostic criteria, ROC and RCS curves showed that the optimal cut-off point for IDAGA was 40, which means a calculated IDAGA ≤ 40 could be used to define PR , B. FurThe average stimulated C-peptide level was much higher in patients during PR [531.8 pmol/L] than that in patients who were not in PR [252.5 pmol/L] (p < 0.001). ROC and RCS curves showed that the corresponding cut-off level for stimulated C-peptide was 244.1 pmol/L (AUC: 0.8) , B for Due to the age-related heterogeneity of clinical features in T1D patients, we explored whether the cut-off value for stimulated C-peptide applied to different age-groups . With GA ≤ 23.0% and insulin dose ≤ 0.5u/kg/day being the diagnostic criteria, the optimal cut-off point for stimulated C-peptide was 265.5, 449.3 and 241.1 pmol/L for the ≤12y, 12-18y and ≥18y age-group, respectively Figure 3High consistency could be observed between different PR definitions , with GA/insulin dose and IDAGA showing the highest consistency, as expected Figure 4We proposed a new definition of PR in T1D using GA and insulin dose, and transformed the definition into a quantitative measure, namely IDAGA. In addition, our study demonstrated that in different age-groups , the cut-off value to diagnose PR was different for stimulated C-peptide, but was relatively consistent for IDAGA. To our knowledge this is the first study to define PR with GA/insulin dose, and come up with the age-dependent cut-off values of stimulated C-peptide for PR diagnosis.As a special stage in T1D, the accurate diagnosis of PR is of particular clinical and research importance. For patients with hyperglycemia onset at older ages, such improved glycemic control should be attached importance to avoid the misdiagnosis of T1D as type 2 diabetes (T2D) . Over haVarious PR definitions have been proposed, but there is no consensus on an easily usable measurement for patients of all ages . In our Due to the age-related heterogeneity in T1D, the cut-off points of IDAGA and stimulated C-peptide were investigated in different age-groups in our study. Since T1D has long been considered a childhood-onset disease, data of T1D in other age-groups are limited, and so does the research related to PR , 11, 29.PR is characterized by the transient recovery of β-cell function and improved glycemic control, and measurements reflecting these two aspects have been be used to define PR . In our Although our results presented a feasible new definition of PR for clinical practice, there are some limitations in our study. First, due to the rather long interval between follow-ups, it was not possible to verify the diagnostic sensitivity with regard of the PR entrance and ending. Second, although age has been shown to correlate with metabolic control in T1D, our definition of PR did not include age. But we took age into consideration during subgroup analysis, and provided the different cut-off values when diagnosing PR in different age-groups. Lastly, we performed the study at a single center with limited number of patients, which may impact its generalizability to other geographical areas or population. Further work is needed to determine whether this definition applies in other racial/ethnic groups.In conclusion, our study proposed a new definition of the PR using GA and insulin dose. The quantitative form of the definition, namely IDAGA ≤ 40, applied to all age-groups. And the study also, for the first time to our knowledge, provided the age-dependent cut-off points of stimulated C-peptide level for PR diagnosis. In addition, it is not recommended to use stimulated C-peptide alone to diagnose PR for patients with signs of insulin resistance.The data analyzed during the current study are available from the corresponding author on reasonable request.The studies involving human participants were reviewed and approved by Medical Ethics Committee of the Second Xiangya Hospital of Central South University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.All authors agreed to submit and publish the manuscript. All authors collected and analyzed data. MS and XJ wrote the first draft of the manuscript. XL designed the study and revised the manuscript. All authors had full access to the data, made final decisions about content, vouch for the accuracy and completeness of the analyses and approved the final version for submission. XL had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.This study was supported by The science and technology innovation Program of Hunan Province (2020RC4044) and Natural Science Foundation of China(8207034059).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Euphorbia tirucalli (E-latex). The nanoparticles (NP) were prepared using the solvent evaporation method and the physical and chemical properties were evaluated using spectrophotometric techniques. FTIR was used to prove the formation of the ester bond between the E-latex and PLGA-NP. The UV-Vis spectroscopic technique was used to show that more than 75% of the latex was encapsulated; the same technique was used to determine the release profile of the compound at different pH values, as well as determining the speed with which the process occurs through kinetic models, and it was observed that the best adjustments occurred for the Korsmeyer-Peppas model and the Higuchi model. The DLS technique was used to determine the diameter of the particles produced as well as their zeta potential (ZP). The sizes of the particles varied from 497 to 764 nm, and it was observed that the increase in E-latex concentration causes a reduction in the diameter of the NP and an increase in the ZP (-1.44 to -22.7 mV), due to more functional groups from latex film being adsorbed to the NPs surfaces. The thermogravimetric experiments exhibit the glass transition temperatures (Tg) that is appropriate for the use of formulated NPs as a stable drug delivery device before use. The in vivo activity of E-NPs (30 and 100 mg/Kg/p.o.) was tested against carrageenan-induced mechanical hypernociception. The data demonstrated a significantly antinociceptive effect for E-NPs, suggesting that E-latex nanoencapsulation preserved its desired properties.The encapsulation of drugs in micro and nanocarriers has helped to resolve mechanisms of cellular resistance and decrease drug side effects as well. In this study, poly (PLGA) was used to encapsulate the Euphol active substance-containing latex from  Several uses of Euphorbia latex (E-latex) include as an antitumor , immunod2 and H2O via the Krebs cycle [Nanocarriers used for medical applications must be biocompatible, able to integrate with a biological system without eliciting an immune response or any negative effects and they must be nontoxic  –18. In tbs cycle –23. Thusbs cycle .® . Distilled water of Milli-Q quality was used. The Euphorbia Tirucalli latex was extracted in our laboratory.Poly , poly , dichloromethane , methyl alcohol anhydrous (99.8%), ethyl alcohol (99.5%), dimethyl sulfoxide anhydrous (99.9%), carrageenan and phosphate buffered saline (PBS) were obtained from Sigma-AldrichEuphorbia tirucalli (E-latex) was collected at 15.8314° S and 48.1142° W at 1170 m altitude . To mitigate component degradation, the latex was immediately transferred to vials and frozen. To improve the quantification and accuracy of the results, the frozen latex was powdered; 12 mL of latex was collected and solubilized in 38 mL of methanol, left to stir for 3 days to obtain the powder and stored in the absence of light [The latex of of light .The analytical curve was made by dissolving 1.0, 2.0, 3.0, 4.0, and 5.0 mg of latex powder in 10 mL of methanol. All solutions (n = 5) had their absorbance values measured spectrophotometrically at a wavelength of 205 nm (Lambda 25 Perkin Elmer spectrophotometer). The obtained results led to the construction of the line equation obtained through linear regression and the analytical curve.Euphorbia tirucalli latex powder. Two solutions were used in the preparation of NPs, the first solution being polyvinyl alcohol (PVA) 2.0%. The second solution was prepared by dissolving 60 mL of dichloromethane (CH2Cl2) in 0.05 g of PLGA [A method for solvent evaporation was chosen, and blank NPs and E-NPs were prepared with the  of PLGA , 27. The of PLGA . PrecipiThe amount of E-latex in the E-NP was calculated using the indirect process, which included separating the supernatants of all preparations, diluting by 100 times, and quantifying using a previously validated calibration curve. The percentage ratio of the amount of drug associated with the nanoparticles to the initial amount of drug used to produce the particles in triplicates was defined as the efficiency of encapsulation using the values obtained from A Zetasizer Nano ZS was used to determine the nanoparticles, size distribution and zeta potentials . The dynamic light scattering mode (DLS) was used to measure the sizes of 100 times diluted E-NPs and blank-NPs. The dispersant used in the study was phosphate-buffered saline (PBS 10 mM). For size analysis, each sample was measured three times with ten runs each time. The Laser Doppler electrophoresis mode was used to determine the zeta potential of each sample, with at least 10 runs at a constant temperature (25°C). Three independent tests yielded size average and zeta potential. Unless otherwise mentioned, all DLS sizes, PdI, and zeta potentials were calculated after dilution .®) was used to obtain spectra for E-latex, blank-NPs, and E-NPs. For the FTIR assay, each sample was first combined with potassium bromide (KBr) at a ratio of 1:10 mg and compressed into a tablet. The detection wavelength was between 400 and 4000 cm-1, and there were 40 scans at a resolution of 4.0 cm-1 [A Fourier Transform Infrared (FTIR) spectrophotometer, model IR Prestige 21 (Shimadzu4.0 cm-1 .® model DSC-60A) using aluminum crucibles with about 3.0 mg of samples, which were subsequently sealed with a press. The analyses were performed under a dynamic nitrogen atmosphere at 20 mL×min-1 and heating rate of 5.0°C×min-1, in the temperature range of 35 to 400°C. The DSC equipment was pre-calibrated with metal indium . The data obtained from the thermal events of the samples were identified in the curves obtained by the software TA-60WS®.The physical state of the E-latex inside the NPs was characterized by the analysis of the DSC curves. The curves were obtained in a DSC cell . In addition, the Korsmeyer-Peppas model is employed to characterize drug release mechanisms: Fickian release (diffusion-controlled release), non-Fickian release  and case-II transport (relaxation-controlled release). When n ≤0.43, it is Fickian release. An n value between 0.43 and 0.85 is defined as non-Fickian release. When n ≥ 0.85, it is case-II transport [Ti is the lag time between the initial of measurement and the release of the drug (in most cases Ti = 0), a is the time scale of the process and b is the shape parameter  [At 37°C the 10 mg of E-NPs was dispersed in 3.0 mL PBS  and UV-Vis absorption values at 205 nm were measured at predetermined time intervals until the release plateau was reached (t = 6 hours). For the kinetic experiments, KinetDS 3.0 software was used; the equations were chosen from the most popular mechanistic and empirical models applied to the drug release curve such as: zero-, first-, second- and third-order, the kinetic models of Higuchi, Korsmeyer-Peppas, Weibull, Hixson-Crowell and the Hill equation , 32.Zeransport . In the f b > 1) .The experiments were performed on male Wistar rats (150–300 g) housed in standard clear plastic cages (four per cage) with free access to food and water. All behavioral testing was performed between 9:00 am and 5:00 pm in a temperature-controlled room. Animal care and handling procedures were in accordance with the International Association for Study of Pain (IASP) Guidelines , the GuiMechanical hypernociception was evaluated in rats using Dynamic Plantar Aesthesiometer (Ugo Basile) . This pr-1), E-NP  and carrageenan (100 μg) were derived from previous studies [Carrageenan was administered locally  at a volume of 100 μL per paw; E-latex or E-NPs were delivered orally (p.o.). The E-latex or E-NP pre-treatment effects (60 min before) were tested against carrageenan-induced mechanical sensitization. The doses of E-latex . p < 0.05 was considered statistically significant.The results are expressed as standard error of the mean (SEM) of measurements made on 5 animals in each group and represent the intensity of mechanical hypernociception. Comparisons across three or more treatments were made using one-way ANOVA with post hoc Tukey E-NP was prepared using the solvent evaporation process because this method is most efficient with drugs which are either insoluble or poorly soluble in the aqueous medium which comprises the continuous phase. The basic characteristics of the NPs prepared for this technique are presented in in vivo distribution, particle stability, etc. In our study, the NPs displayed a mean diameter of between 497 and 764 nm. When administered intravenously, the NPs may have major limitations due to clearance by the reticuloendothelial system (RES). When the particle size exceeds 100 nm, the pharmacokinetic and biodistribution properties greatly change and they are detected in blood and organs like the spleen, lungs, liver, and kidney [NPs applications are predominantly governed by their properties whereby particle size and size distribution are crucial as the size can easily influence the drug loading, release, toxicity, d kidney .Typically, optimal release profiles are achieved by using microspheres with diameters in the range 10–200 μm. For particle diameters < 10 μm, there is a risk that microspheres will be phagocytosed by immune cells. On the other hand, microspheres >200 μm may cause an immune response and inflammation .In addition to intravenous administration, there are other routes of administration that include oral administration, inhalation, intravenous injection, subcutaneous injection, intramuscular injection or in situ humoral injection, to deliver drug-loaded particles to target lesions, thus improving treatment and prognosis . Thus, iThe polydispersity index (PdI), which is a ratio that gives information about the homogeneity of the particle distribution in each system, reflects the quality dispersion within the range of 0.0–1.0. PdI values ≤ 0.1 indicate the highest quality of dispersion. Most researchers recognize PdI values ≤0.3 as optimum values; however, values ≤0.5 are also acceptable . AccordiZeta potential (ZP) is a physicochemical parameter that expresses the stability of a nanomaterial. Extremely positive or negative ZP values cause large repulsive forces, whereas repulsion between particles with similar electric charge prevents aggregation, and accordingly ensures easy redispersion , 41. DifE-latex was entrapment with meaningful statistical significance as E-latex increased from 75 to 78%. Similar results were observed by Khaira et al.  in a stu-1, absence of hydroxyl (υOH) stretching vibrations of Euphol latex, which distinguishes it from the PLGA molecule. The FTIR spectra generated by E-latex, PLGA and E-NPs are represented in FTIR is an important spectroscopic technique for chemical analysis of surface-modified NPs, revealing possible surface interactions. Our knowledge of Euphol presented in latex indicates that it belongs to the class of organic compounds known as triterpenoids, containing six isoprene units  and that−1 represent the presence of–OH and–COOH groups of PLGA. The peaks at 3121, 3004, 2964, 2878 and 2816 cm−1 correspond to–C-H stretching vibrations. The peak at 1753 cm−1 indicates the existence of C = O stretch (Ester), the two peaks at 1636 and 1657 cm-1 are from the Euphol latex which can be attributed to stretching -C = C- and C-C, the signals are slightly shifted to 1623 and 1654 cm-1, after the encapsulation process. The peaks between 1500 and 1250 cm-1 indicate symmetric angular deformation of the CH3 and CH2 functional groups. Between 1500 and 1250 cm-1 CH3 and CH2 symmetric angular deformation predominates, between 1350 and 1150 cm-1 asymmetric angular deformations are present from the CH2 and CH groups.DSC is a powerful tool for the analysis of polymer-drug interactions and has previously been used to show that the drug and polymer are molecularly dispersed . PLGA poin vitro drug release study was carried out for a period of 6.0 h at pH 1.0, 7.4 and 8.5, until the concentration of the E-latex released in the solution reached saturation , as shown in At highly acidic conditions, it was observed that approximately 91% of the compound was released after 45 minutes; after this time E-latex reach out the polymer matrix slowly and sustainably, plateauing after 130 minutes.This is attributed to the process of erosion of the matrix and subsequent release of the drug. PLGA degradation occurs through a process of autocatalytic hydrolysis of the ester bonds in the polymer chains due to protonation of its carboxylic acid group (pKa = 3.85) , 52. EacAs may be observed through In the studies performed at physiological pH 7.4 (green dots) profile it was observed that the best mathematical fit (red line) to the release profile was the two-phase model which can be seen in in vitro release study revealed that at alkaline conditions, 62% of the E-latex is released in the first 105 minutes. In the time interval between 105 and 175 minutes, no significant variation, with approximately 3.0% release, is observed, which can be characterized as a plateau; in the sequence, a sustained release with a few experimental oscillations was observed because of its slow diffusion. After 1.0 h, the amount of E-latex released from the NPs was significantly higher at pH 1.0 than at pH 7.4 and pH 8.5. The amount of E-latex released from the matrix at 1.0 h was estimated to be 92.5% at pH 1.0, 37.7% at pH 7.4, and 39.6% at pH 8.5, indicating that E-NPs followed pH-dependent release kinetics [2 = 0.9922 and a value of n = 0.6294 ± 0.0069 this result and the value of n were determined by plotting log  versus log t [At pH 7.4, a quick release is observed in the first 80 minutes, which is attributed to release of E-latex adhering release to the surface of the NPs. The initial burst is followed by a plateau in a short time of between 85 and 105 minutes, then the release of E-latex follows zero-order kinetics up to 335 minutes when 98.4% of the compound has been released; in the last 25 minutes of monitoring, a plateau is observed. The kinetics , 57. In n that better adjusts to the release profile of an active agent in a matrix system, it is possible to establish a classification, according to the type of observed behavior. Peppas et al. used this n value to characterize different release mechanisms: Fickian diffusion (n = 0.43), Anomalous transport (0.43 < n < 0.85), Case I transport (n = 0.85), Super Case II transport (n > 0.85) [n values were above 0.43  namely, 0.6294 ± 0.0069, suggesting a coupling of diffusion and erosion mechanisms, anomalous diffusion, and possibly indicating that Euphol latex release from E-NPs was controlled by more than one process. The results obtained are consistent with those described in the literature [This model is generally used to analyze the release of pharmaceutical polymeric dosage forms when the release mechanism is not well-known or when more than one type of release phenomenon could be involved . It is i > 0.85) , 58. In terature , 32, 59,At alkaline pH 8.5 the release profile of Euphol latex also showed a biphasic behavior (black dots) as shown in in vivo for an ester end-capped PLGA in comparison with a more hydrophilic acid-terminated PLGA of a similar molecular weight and co-polymer composition [2 = 0.9877.Under neutral or alkaline conditions, PLGA can undergo several terminal modifications, such as favoring the conversion of the free carboxylic acid terminal group to an ester terminus, which considerably affects the final physicochemical characteristics. It is reported in the literature that the mucoadhesive properties of PLGA increase the residence time of the drug-laden delivery system, improving its oral absorption . A delayposition . As showAs described before, isolated Euphol latex showed an antinociceptive effect on mice after oral administration , 60, 61.The comparison between both E-latex and E-NP intensity of effects were shown to be significantly different. Once administered, drug release is an important parameter, to know how the system will maintain the concentration of the compound in the target tissues at a desired value as long as possible, exerting a control on the drug release rate and duration. The effect of pH on the release profile was observed, mimicking the release in gastrointestinal fluid (pH 1.0), in the small intestine and blood (pH 7.4) and in the colon (pH 8.5). The results indicate that the E-NPs followed pH-dependent release kinetics with a quick exit of the compound under acidic conditions. Pharmacokinetic as well pharmacodynamic properties conferred by nanoencapsulation of E-latex can be explored in future studies. The data demonstrate the antinociceptive effect for E-NP, suggesting that E-latex nanoencapsulation preserved its desired properties The The results of this study revealed that E-NPs were prepared successfully using the solvent evaporation method with an encapsulation efficiency of ≥75%. FTIR demonstrated the polymer and Euphol latex interaction. The physicochemical characteristics of this release system such as hydrodynamic diameter, zeta potential and physical stability up to 37°C, as well as the biocompatibility and biodegradability of the polymer indicate that E-NPs are suitable for oral delivery due to versatile biodegradation in distinct pH. Animal assays with E-NPs or E-latex significantly inhibited the mechanical hypernociceptive induced by carrageenan, suggesting that the nanoencapsulation process of Euphol latex is impaired during the entrapment and administration process. Further studies are needed to explore the use of nanotechnology for the possible antinociceptive effect induced by E-latex.


Mycobacterium kansasii pulmonary disease. We retrospectively collected clinical data from 33 patients with M. kansasii pulmonary disease to determine the time-to-sputum culture conversion (SCC) upon treatment with a standard combination regimen consist of isoniazid-rifampin-ethambutol. Next, MIC experiments with 20 clinical isolates were performed, followed by a dose-response study with the standard laboratory strain using the hollow-fiber system model of M. kansasii infection (HFS-Mkn). The inhibitory sigmoid maximum effect (Emax) model was used to describe the relationship between the bacterial burden and rifampin concentrations. Finally, in silico clinical trial simulations were performed to determine the clinical dose to achieve the optimal rifampin exposure in patients. The SCC rate in patients treated with combination regimen containing rifampin at 10 mg/kg of body weight/day was 73%, the mean time to SSC was 108 days, and the mean duration of therapy was 382 days. The MIC of the M. kansasii laboratory strain was 0.125 mg/L, whereas the MICs of the clinical isolates ranged between 0.5 and 4 mg/L. In the HFS-Mkn model, a maximum kill (Emax) of 7.82 log10 CFU/mL was recorded on study day 21. The effective concentration mediating 80% of the Emax (EC80) was calculated as the ratio of the maximum concentration of drug in serum for the free, unbound fraction (fCmax) to MIC of 34.22. The target attainment probability of the standard 10-mg/kg/day dose fell below 90% even at the MIC of 0.0625 mg/L. Despite the initial kill, there was M. kansasii regrowth with the standard rifampin dose in the HFS-Mkn model. Doses higher than 10 mg/kg/day, in combination with other drugs, need to be evaluated for better treatment outcome.There is limited high-quality evidence to guide the optimal treatment of  Mycobacterium kansasii is one of the most virulent and prevalent NTM species and one of the six most frequently isolated NTM species across the world. The clinical presentation of M. kansasii pulmonary disease mimics classical tuberculosis (TB) diseases caused by Mycobacterium tuberculosis  are a growing burden on a global level, with an increasing number of mycobacterial species described  and a grrculosis .M. kansasii pulmonary disease is a combination regimen extrapolated from TB treatment. The drugs in the standard combination regimen are isoniazid (INH) (5 mg/kg of body weight/day), rifampin (10 mg/kg of body weight/day), and ethambutol (15 mg/kg of body weight/day). The recent multisociety NTM treatment guideline recommends daily or intermittent therapy when a macrolide-based regimen is used and daily therapy when an INH-based regimen is used  studies conducted to optimize the dose of rifampin as well as other drugs in the combination therapy for the treatment of M. kansasii pulmonary disease. There are also no randomized control trials that have been conducted to associate the baseline MIC of the infecting strains with the clinical outcome.Rifampin is one of the key drugs in the  failure , 6. One rifampin . Unlike M. kansasii infection is classified as a rare disease, which makes it difficult to establish an evidence base for therapeutic decision-making (https://rarediseases.info.nih.gov/diseases). Therefore, to fill in the knowledge gap on the optimal dose of the drugs in the standard regimen, it is essential to perform monotherapy PK/PD experiments to determine the optimal dose of each drug, followed by experiments comparing the efficacy of the optimal dose combination with the standard of care regimen for efficacy and resistance suppression. Therefore, the aim of the present study was (i) to determine the time to negative sputum culture conversion (SCC) and therapy duration in patients treated with the standard combination regimen for M. kansasii pulmonary disease, (ii) to perform rifampin susceptibility testing and determine MICs of 20 M. kansasii clinical strains, (iii) to perform PK/PD studies using the preclinical hollow-fiber system model (–M. kansasii infection (HFS-Mkn) to determine the optimal rifampin exposure for M. kansasii kill, and (iv) to determine the target attainment probability (TAPs) of different clinical doses of rifampin to achieve the optimal exposure target, as identified in the HFS-Mkn model, across the MIC range in clinical strains. We propose that the rifampin optimal dose combination regimen will result in successful treatment outcome and also could possibly shorten the therapy duration for M. kansasii pulmonary disease.Pulmonary em model –10 of M.M. kansasii pulmonary disease and treated with the standard combination regimen  and who had serial sputum samples collected during the therapy. The baseline patient demographics, clinical characteristics, sputum culture conversion (SCC), and therapy duration are summarized in We used the data from a retrospective study to benchmark the efficacy of the standard regimen containing 10 mg/kg/day rifampin. There were 33 patients fulfilling the criteria for 50 and MIC90 were calculated as 0.5 and 4 mg/L, respectively. Emax) model was used to describe the relationship between the bacterial burden and drug concentration (expressed as “×MIC”). Compared to the nontreated controls, rifampin killed 5.00 ± 0.34 log10 CFU/mL M. kansasii in 7 days. The rifampin concentration mediating 50% of the maximal kill Emax (EC50) was calculated as 0.88×MIC, or 0.11 mg/L.The rifampin MIC of the laboratory strain was 0.125 mg/L. Mkn study are shown in Mkn study, the rifampin half-life was calculated as 6.09 h , which was indeed longer than the intended 3-h half-life; however, it was still in the range of that reported in clinical trials  , and the volume of distribution was calculated as 285.8 L . Mkn model’s measured drug concentrations. The measured drug concentrations were used to calculate ratio of the the maximum concentration of drug in serum for the free, unbound fraction of drug (fCmax) to MIC and area under the concentration-time curve from 0 to 24 h for the free, unbound fraction of drug (fAUC0–24)/MIC with each of the eight rifampin doses, as summarized in The concentration-time profiles of different rifampin doses in the HFS-10.45 h) . Among tMkn model over the 28-day study period are shown in Fig. S1 in the supplemental material. Mkn model over 28 days of the study. The extent of bacterial kill was dose dependent, and the maximum kill of 7.81 log10 CFU/mL compared to the nontreated controls was recorded on day 21 of the study. The relationships between the CFU and rifampin concentration (fCmax/MIC ratio and fAUC0–24/MIC ratio) are shown in r2 value and lowest Akaike information criterion score (AICc)   were use50 was calculated as an fCmax/MIC ratio of 3.53 with an H of 0.61 (r2 = 0.99). This translates to an EC80 or optimal fCmax/MIC ratio for M. kansasii kill as 34.22. Regarding the emergence of rifampin resistance in the HFS-Mkn model, although we noticed the change in the kill curve trajectories on study day 28, there was no rifampin-resistant subpopulation recorded on agar supplemented with 3 mg/L rifampin. Since, 3 mg/L corresponds to 24×MIC of the ATCC strain used in the HFS-Mkn study, we performed a MIC experiment using the day-28 MGIT-positive cultures . There was no change in the rifampin MIC in the nontreated control systems as well as in the culture from HFS-Mkn model treated with the different rifampin. The results of the MGIT-derived time-to-positive (TTP) readouts, as the second pharmacodynamics measure, are shown in Fig. S3 in the supplemental material.On day 21 when the maximal kill was recorded, the rifampin ECfCmax/MIC ratio of 34.22 was simulated using the input population PK data from the literature (The target attainment probability (TAP) of different rifampin clinical doses, ranging from 10 mg/kg  to 40 mg/kg  to achieve the optimal exposure target of an terature . Figure M. kansasii pulmonary disease, where rifampin is one of the key drugs. While prior studies show high failure rates of the combination regimen  was only 73%, the mean time to SCC was 108 days, and the mean duration of therapy was 382 days. While only 33 patients were included in the study, these findings still reinforce that higher-than-recommended standard doses of the drugs, including rifampin, are required for a successful treatment outcome and lend credence to the aim that a higher dose combination may lead to shorter duration of therapy than the currently recommended 12 months  of the rifampin-containing standard combination regimen for the treatment of 2 months .M. tuberculosis studies  of the agar-based culture methods. It could also be that we used a single phenotypic method for resistance determination and did not assay the rpoB resistance-determining region, where mutations may have been observed in the absence of phenotypic resistance .To benchmark the efficacy of currently recommended treatment regimen, we performed a retrospective study to collect clinical data from a subset from a previously published study  with Tai Society . Serial  Society , 22. HosM. kansasii standard laboratory strain (ATCC 12478) and 20 clinical strains (separate from the 33 patients in the retrospective study) were used in the experiments. Middlebrook 7H9 broth and Middlebrook 7H10 agar, both supplemented with 10% oleic acid-albumin-catalase-dextrose (OADC), were used to culture the bacteria , polyvinylidene difluoride (PVDF) hollow-fiber cartridges were purchased from FiberCell Systems, Inc. , and the MGIT liquid culture system and EpiCenter software were purchased from Becton Dickinson, USA.The ATCC 1247 and 20 c5 CFU/mL. Next, 180 μL of the inoculum was added to each of the 96 wells prefilled with 20 μL of each rifampin concentration (10×), ranging between 0.125 and 64 mg/L. After 7 days of incubation at 37°C, cultures were visually inspected using an inverted mirror. The lowest rifampin concentration that completely inhibited M. kansasii growth  was determined as the MIC.The MIC of rifampin was determined using the broth microdilution method . The MICM. kansasii colonies were counted after 10 days of incubation at 37°C. The relationship between the different rifampin concentrations and the M. kansasii bacterial burden (log10 CFU/mL) was described using the inhibitory sigmoid Emax model.The experiment was performed only with the standard laboratory strain ATCC 12478, with inoculum preparation and drug concentration range as described above. The total culture volume was 5 mL. After 7 days of drug exposure, a 1-mL sample from each test tube was collected, washed twice to remove carryover drug, and serially diluted 10-fold before inoculation on Middlebrook 7H10 agar supplemented with 10% OADC. The M. kansasii (Mkn model was used without any modification. The dilution rate in the HFS-Mkn model was set to mimic the rifampin pharmacokinetics achieved in patients (f) drug concentration to exert the antimycobacterial effect is 20% of the total rifampin concentration  in HIV-negative patients (0–24 was 62.61 + 2.44 mg · h/L (fAUC0-24 12.55 ± 0.08) in HIV-infected patients. Thus, since it is the free or non-protein-bound fraction of a drug that is pharmacologically active  of 1:1. After 4 h of infection, THP-1 cells were washed twice with warm RPMI 1640 to remove the extracellular bacteria. Next, 20 mL of the infected THP-1 cells was inoculated into the peripheral compartment of each of the 10 HFS-Mkn units. The circulating medium was RPMI 1640 supplemented with 2% FBS. The HFS-Mkn units were treated with different rifampin doses, where the drug was administered over 1 h into the central compartment. The drug infusion rate and time were controlled using computerized syringe pumps, and 1st order kinetics was used to explain the drug diffusion into the peripheral compartment (across the hollow fibers into and out of the extracapillary space). To study the steady-state PK of each rifampin dose, on day 7 of the study, the central compartment of each HFS-Mkn unit was sampled predose, followed by 1, 3, 6, 9, 12, and 23.5 h post-drug infusion. For the pharmacodynamics of rifampin in the HFS-Mkn model, samples were collected from the peripheral compartment of each HFS-Mkn unit on days 3, 7, 10, 14, 21, and 28. The number of viable THP-1 cells in each HFS-Mkn unit was determined using an automated cell counter  as well as by manual counting with a hemocytometer. The carryover drug in the samples was removed by washing twice with normal saline, followed by 10-fold serial dilution to determine the bacterial burden using the Middlebrook 7H10 agar supplemented with 10% OADC. The same samples were also cultured on agar supplemented with 3 mg/L rifampin to determine the drug-resistant subpopulation at each time point. The CFU were recorded after 10 days of incubation at 37°C. As a second pharmacodynamics method, we used the MGIT liquid culture system, because it is more sensitive than the solid agar culture methods. Five hundred microliters of the washed and undiluted sample from each HFS-Mkn unit was inoculated into the MGIT tubes. EpiCenter software was used to record the growth units and time to positive in each sample.To briefly describe the infection of the HFS-In addition, on day 28, we performed MIC experiments with bacteria grown in the MGIT tubes, to determine the actual change in the MIC using a colorimetric resazurin assay (m/z 823 to 791 for rifampin and m/z 826 to 794 for rifampin-d3. The between-day percentage of coefficient of variation (%CV) for analysis of low-quality controls  controls were 8% (8%). The intraday %CV for rifampin was 8% (6%). The lower limit of quantitation was 0.01 μg/mL.The method to measure the rifampin concentration in the experimental samples was published previously and was used without any modification , 33, 34.Mkn model were modeled using a one-compartment model with first-order input and elimination and used to calculate the peak concentration (Cmax) and AUC0–24, as well as the half-life, clearance rate, and volume of distribution, by using Phoenix WinNonLin . The exponential-growth model was used on the MGIT-derived time-to-positive readouts . We used GraphPad Prism (v8) for graphing the data.Measured rifampin concentrations in the HFS-inNonLin . The fou80 concentration, we performed clinical trial simulations with 10,000 virtual patients. The population PK parameter input estimates were those identified by Wilkins et al. (−1 (IIV = 2.39)  . The rif = 2.39) , 30. Thev et al. .


Plywood is made of wood veneers that are bonded with adhesives such as urea-formaldehyde, phenol-formaldehyde and melamine-formaldehyde resins. The plywood made from formaldehyde-based adhesives not only releases formaldehyde but also relies on fossil resources. In this article, we synthesized furan-acetone adducts from lignocellulosic biomass in one pot. The furan-acetone adducts could be directly used as adhesives with the addition of phosphoric acid as a curing catalyst. Particularly, with the addition of 5 wt% diphenylmethane diisocyanate (MDI) as a crosslinking agent, both the wet and dry bonding strength of the plywood prepared from the adhesives could meet the minimum requirement of 0.7 MPa . The possible adhesion mechanism is that the penetration of furan-acetone adhesives into vessels and cell lumens followed by crosslinking during hot-pressing forms mechanical interlocking at the interface of wood veneers, which provides the main bonding strength of plywood. The findings presented here could provide a new way for the efficient preparation of aldehyde-free green wood adhesives and the value-added utilization of woody biomass. Wood-based panels such as plywood, particleboard, and fiberboard, are often used in furniture and architectural decoration . They arCellulose and hemicellulose derivatives such as furfural and 5-hydroxymethyl (HMF) products could self-condense into humins, but their high permeability into wood cells and water solubility prevents them from forming robust cross-linked adhesives . InsteadWe believed that the crosslinking ability of furfural-acetone could allow it to be a potential adhesive to replace synthetic resins in the field of wood-based panel production. In the biorefining area, research on the preparation of furan-acetone adducts from biomass-derived platform molecules  has mainly focused on diesel and jet fuel production . Its synThe cost-effective conversion of polysaccharides in lignocelluloses to HMF and furfural remains an issue since they tend to further condense to form insoluble humins in an acidic aqueous solution . Therefop-toluenesulfonic acid (99%) were purchased from Innochem . Dichloromethane (99%), lithium bromide (99%), furfural (99%), and phosphoric acid (75 wt%~80 wt%) were purchased from Aladdin® Chemicals ; hydrochloric acid (37 wt%), acetone (99%), and ethanol (99%) were purchased from China National Pharmaceutical Group Corporation . 5-hydroxymethylfurfural (HMF) was purchased from Bidepharm . Eucalyptus and masson pine (40–80 mesh) were provided by Qingshan Paper Co., Ltd. , and the straw (40–80 mesh) was obtained from the State Key Laboratory of Bio-based Materials and Green Paper of Qilu University of Technology .All reagents were analytically pure and were used immediately upon receipt. Glucose (99%), xylose (99%), tetrahydrofuran (99%), pyridine (99%), glutaraldehyde (50%), epichlorohydrin (98%), hexanediamine (99%), diphenylmethane diisocyanate (98%), and Glucose-based furan-acetone adducts (GluA): glucose (8 g), 8 mL deionized water, 24 g LiBr, 80 mL acetone, and 0.44 mL hydrochloric acid (37 wt%) were added to a 350 mL thick-walled pressure-resistant vessel. After the vessel was sealed, the vessel was heated in an oil bath at 120 °C for 1 h. After the reaction was completed, the residual acetone in the reaction solution was removed by a rotary evaporator, and the solid residue was extracted with 100 mL dichloromethane. After complete dissolution and extraction, the extract was transferred to a centrifuge tube and centrifuged at 10,000 rpm for 15 min, and the upper dichloromethane phase was separated and dichloromethane was removed by a rotary evaporator to obtain a black solid, GluA.Xylose-based furan-acetone adducts (XylA): The procedure was the same as that for GluA, except that 8 g xylose instead of 8 g glucose was added.Eucalyptus-based furan-acetone adducts (EA): The procedure was the same as that for GluA, except that 8 g of eucalyptus powder (40−80 mesh) and 0.88 mL of hydrochloric acid (37 wt%) instead of 8 g glucose and 0.44 mL of hydrochloric acid (37 wt%) were added.Masson pine furan-acetone adducts (MA): The procedure was the same as that for EA except that 8 g of masson pine wood powder (40−80 mesh) was added.Straw-based furan-acetone adducts (SA): The procedure was the same as that for GluA, except that 8 g of straw powder (40−80 mesh) and 1.02 mL of hydrochloric acid (37 wt%) instead of 8 g glucose and 0.44 mL of hydrochloric acid (37 wt%) were added.Furfural-acetone adducts: An amount of 0.8 g furfural, 8 mL deionized water, 24 g LiBr, 80 mL acetone, and 0.44 mL hydrochloric acid solution (37 wt%) were added to a 150 mL thick-walled pressure-resistant vessel. After the vessel was sealed, the vessel was heated in an oil bath at 100 °C for 0.5 h. After the reaction was completed, the residual acetone in the reaction solution was removed by a rotary evaporator, and the solid residue was extracted with 100 mL dichloromethane. After complete dissolution and extraction, the extract was transferred to a centrifuge tube and centrifuged at 10,000 rpm for 15 min, the upper dichloromethane phase was separated, and dichloromethane was removed by a rotary evaporator to obtain a black solid .HMF-acetone adducts: An amount of 0.8 g HMF, 8 mL deionized water, 24 g LiBr, 80 mL acetone, and 0.44 mL hydrochloric acid solution (37 wt%) were added to a 150 mL thick-walled pressure-resistant vessel. After the vessel was sealed, the vessel was heated in an oil bath at 120 °C for 1 h. After the reaction was completed, the residual acetone in the reaction solution was removed by a rotary evaporator, and the solid residue was extracted with 100 mL dichloromethane. After complete dissolution and extraction, the extract was transferred to a centrifuge tube and centrifuged at 10,000 rpm for 15 min, the upper dichloromethane phase was separated, and dichloromethane was removed by a rotary evaporator to obtain a black solid (HMF-acetone adducts).p-toluenesulfonic acid (p-TsOH) was added as a curing agent, and the loading of the curing agent was about 2–15% of the mass of the adduct. Hexamethylene diamine (EDA), glutaraldehyde (GA), epichlorohydrin (ECH), or diphenylmethane diisocyanate (MDI) was added as cross-linking agents during the preparation process, and the amount of the added cross-linking agent was about 3–9% of the adhesive mass. After the addition of all additives, the mixture was fully stirred and then coated on the surface of the veneers.Preparation of adhesive: Initially, the adducts were vacuum dried at 50 °C for 24 h and then ground into a powder with a quartz mortar. About 10–20 g of the adducts was weighed with the addition of acetone (about 50% mass of the adduct) for dissolving the adduct. The mixture was stirred evenly to form a gel. Next, hydrochloric acid (HCl), phosphoric acid (PA), or 2. Prior to hot-pressing, the coated wood veneers were placed in an oven and treated at 80 °C for 8 h to remove excess acetone and prevent glue penetration. Then the three-layer poplar plywood was pressed by a laboratory-scale hot-presser  at 120–200 °C for 5–20 min.Preparation of three-layer poplar plywood: The poplar veneer was cut into several sheets with a size of 100 mm × 300 mm × 1.6 mm, and the moisture content of the veneers were between 8% and 15%. The grain of each layer was perpendicularly oriented with its adjacent layer. The adhesives were evenly coated on the two sides of the middle-layer veneer  by a rubGas chromatography-mass spectrometry (GC-MS): Ten mg of adduct was dissolved in 1 mL of chloroform in a GC vial. GC-MS analysis was performed using the following temperature program: 40 °C, 8 min; 40–300 °C, 10 °C/min; and 300 °C, 5 min. The chromatographic conditions were as follows: a Techcomp SCION-5MS  capillary column with high-purity helium as carrier gas was used, and the injection volume, split ratio, and solvent delay time were fixed as 1 μL, 15:1, and 8 min, respectively. The mass spectrometry conditions were as folows: the ion source voltage and its temperature were 70 eV and 200 °C, respectively.Mechanical analysis of plywood: The plywood samples were stored in air for at least 24 h before mechanical testing. Lap-shear tests of the three-layer plywood were performed on a tensile machine  with a crosshead speed of 10 mm/min according to the Chinese National Standard GB/T 9846–2015 [Observation of glue lines: Nikon optical microscope  was used to observe the glue lines of the plywood samples. First, slices were cut at the cross-section of the sample using a knife; then, the slices were fixed on a slide; finally, the slide was placed on the optical stage for testing.Scanning electron microscope (SEM) analysis: SEM  was used to analyze the fracture surface of the glue layer and the morphology of the glue lines of the plywood samples. The samples were firstly dried in a vacuum oven at 50 °C for 24 h. Then, the dried samples were adhered to the sample stage with conductive adhesive and sprayed with gold; finally, SEM tests were performed in high vacuum operation mode at 5.0 kV.−1 with 64 scans at a resolution of 4 cm−1 at room temperature.Attenuated Total Reflection-Fourier transform infrared spectroscopy (ATR-FTIR) analysis: ATR-FTIR analysis was carried out on a Nicolet IS 50 spectrometer  over a region of 500–4000 cmThermogravimetric (TG) analysis: A thermogravimetric analyzer  was used to analyze the thermal stability of the cured adhesives. The heating rate was 10 °C/min in a nitrogen atmosphere, the temperature range was 30–800 °C, and the mass of the test sample was 7–10 mg.1 × 40 + n2 × 58  was used to estimate the molecular weights of such self-condensation products  derived from lignocellulosic biomass. Meanwhile, compared with furfural-acetone adduct and HMF-acetone adduct, GluA, XylA, EA, MA, and SA showed the same absorptions at these three wavenumbers, indicating that furan-acetone adducts could be directly produced from carbohydrates and lignocelluloses in one pot. Compared to the acetone-acetone adducts, GluA, EA, MA, and SA presented a wider absorption band at 3415 cm−1, which is a representative of the hydroxyls in the measured samples, indicating the formation of HMF-acetone adducts from these feedstocks. The ATR-FT-IR spectra of furan-acetone adducts combined with the previous GC-MS analysis of these compounds further verified that the furan-acetone compounds were successfully synthesized from lignocellulosic biomass in one pot.ATR-FT-IR was used to further characterize the biomass-derived furan-acetone adduct samples . Comparep-TsOH was between HCl and PA, so its effects on curing adhesive were moderate compared to HCl and PA. Therefore, a medium-strength acid such as PA was suitable for curing furan-acetone adducts at high curing temperatures without deconstructing the structure of wood veneers. With the increase of PA addition  were further investigated. All four biomass-based furan-acetone adducts exhibited similar wet and dry bonding strengths a, which Since the wet strength of the plywood should exceed 0.7 MPa to meet the quality requirement, the effects of several cross-linkers on the strength of plywood prepared from furan-acetone adhesives were analyzed. The cross-linking agents were expected to react with the hydroxyls and/or carbonyls in furan-acetone adducts and hydroxyls on the surface of wood veneers to improve the cross-linking of the furan-acetone adducts and wood-adhesive interface. As shown in Since MDI showed the best promotion effect on the mechanical properties, its addition was further optimized. As shown in −1 and N-H at 1149 cm−1 were attenuated for the five samples, which indicated that MDI was indeed introduced into the adhesives, and MDI reacted with hydroxyl groups to form carbamate bonds during the curing process of the adhesives. In addition, compared with the other four furan-acetone adhesives, GluA contained an additional hydroxyl group in the structure of HMF, enabling a higher capability of reacting with isocyanate groups to form cross-linked network and thereby increasing the adhesive strength of the plywood. Therefore, this observation further explained why the glucose-based furan-acetone adducts showed the highest bonding strength of 1.00 MPa for wet bonding strength and 1.12 MPa for dry bonding strength  . The DTGThe mechanical joining effect, chemical bonding effect, and hydrogen bonding were the main factors influencing the adhesion between the adhesive and the wood veneer. Analysis of the gluing lines of the plywood using an optical microscope showed that the adhesive penetrated the cell lumens and vessels of the poplar wood veneers and filled the cell cavities to form “glue nails” , which eIn conclusion, a variety of carbohydrate-based furan-acetone adduct adhesives from woody biomass were synthesized in a one-pot catalytic reaction system using acetone as a solvent and acidic lithium bromide as a catalyst. This process was carried out under a mild temperature of 120 °C with recyclable catalysts and was able to directly use biomass as feedstock without pretreatment or fractionation. After the optimization of experimental conditions, the best curing effect was obtained with a medium-strength acid, i.e., PA, as a curing catalyst, and the wet and dry bonding strengths could be substantially improved with the addition of crosslinking agents such as MDI. The curing mechanism of furan-acetone adhesives derived from lignocellulosic biomass may mainly involve chemical reactions such as aldol condensation between furan-acetone adducts, the formation of carbamate bonds between side chain hydroxyl groups of furan-acetone adducts, and the hydroxyls on the surface of wood veneers. Based on these observations, the possible adhesion mechanism is that the penetration of furan-acetone adhesives into vessels and cell lumens followed by crosslinking during hot-pressing forms mechanical interlocking at the interface of wood veneers, which provides the main bonding strength of plywood. These sustainable bio-based adhesives can be considered environmentally friendly and cost-effective alternatives to formaldehyde-based adhesives for plywood production. Although carbohydrate-based furan-acetone adhesives could be used to prepare three-layer poplar plywood, the curing conditions of plywood prepared by carbohydrate-based furan-acetone adhesives still need to be optimized to reduce the cost in energy consumption and pressing time compared with those prepared by conventional formaldehyde resin adhesives.